The invention relates to the analysis of biological matter and, more particularly, to the comparison of isotopically labeled components of biological matter from one sample with the same, unlabeled components of biological matter from another sample, through mass spectroscopy. The method is particularly suited for quantifying differences in protein expression or modification in two cell populations or pools, one of which is subjected to environmental, genetic or chemical modulation.
Many biological processes in living cells are controlled by alterations in the levels or states of certain key proteins. Measuring the levels of the various proteins that affect (or are affected by) the process is therefore important for gaining an understanding of the biological process. For example, a given hormone may, through a signaling cascade, activate certain key transcription factors which in turn induce the expression of a number of proteins with distinct activities. Comparison of the levels of the proteins in the cell prior to and after induction can indicate which gene products are being up regulated and/or down regulated by the action of the hormone. As a second example, comparison of the total complement of proteins from an organism (i.e., the proteome) prior to and after infection with a virus can show which proteins are down and/or up regulated by the infection. Such an analysis can provide important information about the mechanism by which the virus subverts its host cell, thereby aiding in the development of anti-viral drug strategies. Similarly, comparison of some or all of the proteins of the proteome before and after treatment with a drug can indicate the mechanism of action of the drug, as well as its potential effectiveness and toxicity. As another example, measurement of the state of phosphorylation of protein members of an intracellular cascade involved in turning on and off a given biological process can provide information about the control of the signaling pathway.
A facile method for accurately comparing the levels of proteins and other cellular components and biological materials as a function of time or as the result of particular treatment, such as a hormone, a drug, or a virus, as mentioned above, or an environmental stimulus, such as a temperature change, is needed. It is also necessary to assay these protein levels with high accuracy because small changes in the levels of certain key proteins may, through a complex cascade of molecular events, produce large changes in the biological system.
Two-dimensional electrophoresis has been used to compare proteins from different cell cultures or hosts subjected to differing conditions. See, for example, Anderson, N. G., et al., xe2x80x9cSimultaneous Measurement of Hundreds of Liver Proteins: Application in Assessment of Liver Function,xe2x80x9d Toxicologic Pathology, 1996, Vol. 24, No. 1, pp. 72-76; Anderson, N. G., et al., xe2x80x9cTwenty years of two-dimensional electrophoresis: Past, present and future,xe2x80x9d Electrophoresis, 1996, Vol. 17, pp. 443-453; Anderson, N. G., xe2x80x9cCovalent Protein Modifications and Gene Expression Changes in Rodent Liver Following Administration of Methypyriline: A Study Using Two-Dimensional Electrophoresis,xe2x80x9d Fundamental and Applied Toxicology, 1992, Vol. 18, pp. 570-580; and Anderson, N. G., et al., xe2x80x9cGlobal Approaches to Quantitative Analysis of Gene-Expression Patterns Observed by use of Two-Dimensional Gel Electrophoresis,xe2x80x9d Clin. Chem. 1984, Vol. 30, No. 12, pp. 2031-2036.
FIG. 1 is a schematic representation of the processes described in these articles. Proteins from a control cell culture are extracted, purified and separated by one- and two-dimensional electrophoresis. Proteins from another, parallel cell culture, which may include cells exposed to drugs, carcinogens or other such treatments directly or through a host, are also extracted, purified and separated by one- and two-dimensional electrophoresis. FIG. 2 shows exemplary electrophoretic gel samples from each cell culture. Spots at different locations in each gel sample may indicate the presence of different proteins or changes in the proteins in the control versus the treated cells. Spots of different sizes may indicate a change in the quantity of the protein in the treated cells. The gels may be analyzed visually or by labeled maps, bargraphs or numerical tables. See, Anderson, xe2x80x9cTwenty years of two-dimensional electrophoresis . . .xe2x80x9d, at p. 450. Computer generated arrowplots, which indicate the magnitude and polarity of changes in spots between gel samples of a control and treated cell pool, superimposed on a gel sample of a control cell pool, have also been used. Id. Instead of a control cell sample, the gel sample including the proteins from the treated cells may be compared to a master gel pattern from a library of gel patterns.
Using prior art methods, hundreds of gel samples and hundreds of thousands of protein abundance measurements may be required in a typical study. Id. It is also difficult to maintain the reproducibility of the extraction and purification procedures in each cell sample. Extraction and purification results must be normalized. Precise, accurate and reproducible quantification of the changes between cell pools is also difficult. If a gel spot includes more than one protein, the discrete proteins frequently cannot be identified. Thus, a more practical method of comparing proteins in different cell pools is needed.
Mass spectroscopy is a highly accurate analytical tool for determining molecular weights and identifying chemical structures. Proteins and peptides have been studied by matrix-assisted laser desorption mass spectroscopy and electrospray ionization mass spectroscopy. See, for example, Chait, Brian T. and Kent, Stephen B. H., xe2x80x9cWeighing Naked Proteins: Practical, High-Accuracy Mass Measurement of Peptides and Proteinsxe2x80x9d, Science, Vol. 257, Sep. 25, 1992, pp. 1885-1894, which is incorporated by reference herein. Matrix-assisted laser desorption time-of-flight mass spectrometers are described in U.S. Pat. Nos. 5,045,694 and 5,453,247, to Beavis, et al., which are assigned to the assignee of the present invention and incorporated by reference herein. Electrospray ionization mass spectrometers are described in U.S. Pat. No. 5,245,186 to Chait et al., and U.S. Pat. No. 4,977,320 to Chowdhury et al., for example, which are also assigned to the assignee of the present invention and incorporated by reference herein. Prior to analysis, the proteins are typically separated by one- or two-dimensional electrophoresis and then digested by an appropriate enzyme. The resulting peptides are then subjected to mass spectroscopy by any of the types of mass spectrometers identified above.
However, quantitative comparisons among proteins within a sample or between samples may be compromised by a number of parameters, such as the ionization efficiency of the mass spectrometer for a particular peptide or protein, the extraction efficiency from electrophoretic gels for a particular peptide and the digestion efficiency of an enzyme at different cleavage sites.
Isotopic labeling by stable or radioactive isotopes has been used to study many aspects of human, animal and plant metabolism. For example, isotopic labeling has been used to study metabolic turnover rates and biosynthesis of proteins and nucleic acids. Microorganisms, organs and tissue extracts, for example, may also be studied through isotopic labeling. The presence of radioactive isotopes in a sample of biological material may be detected by scintillation counters, or autoradiography, for example. However, the use of radioactive isotopes pose hazards to those conducting the experiments and require the use of protective measures, which may be cumbersome and expensive. To avoid this problem, in U.S. Pat. No. 5,366,721, a long-lived radioisotope, such as carbon-14, is administered to a biological host. A reacted fraction is isolated from the host and the radioisotope concentration is measured by mass spectroscopy. See also DeLeecher, A. P. et al., xe2x80x9cApplications of isotope dilutionxe2x80x94mass spectrometry in clinical chemistry, pharmacokinetics, and toxicology,xe2x80x9d Mass Spectroscopy Reviews, 1992 11, 249-307; Grostic, M. F. et al., xe2x80x9cMass-Spectral Studies Employing Stable Isotopes in Chemistry and Biology,xe2x80x9d appearing in Mass Spectroscopy: Techniques and Applications, edited by Mike, G. W. A., Wily-Interscience (1971), pp. 217-287.
The present invention is a method for accurately comparing the levels of ionizable components of biological matter, wherein the biological matter differs in some respect from each other, using mass spectroscopy and isotopic labeling.
In one embodiment of the present invention, a method for comparing the relative abundance of a protein of interest in multiple samples of biological matter is disclosed, wherein one of the samples has been modulated by exposure to a treatment, such as a bacteria, virus, drug or hormone, or a stimulus, such as a chemical or environmental stimulus. A first sample of the biological matter is cultured in a first medium containing a natural abundance of isotopes and a second sample of the biological matter is cultured in a second medium containing more or less than the natural abundance of one or more isotopes. One of the samples is modulated, at least portions of the samples are combined and at least one protein is removed from the combined sample.
The removed protein, which may or may not be digested into peptides, is subjected to mass spectroscopy to develop a mass spectrum. The difference in the mass of the isotope in each cell pool results in two distinct, closely spaced peaks for each protein or peptide in the mass spectrum. One peak corresponds to a protein or peptide from a protein from the cell pool with the normal abundance of isotopes. The other peak corresponds to a protein or peptide from the cell pool enriched in one or more of the isotopes. A ratio is computed between the peak intensities of at least one pair of peaks in the mass spectrum. The relative abundance of the protein in each sample may be determined based on the computed ratio. The protein may be identified by the mass-to-charge ratios of the peaks in the mass spectrum, as well as by other means known in the art.
In addition, modifications, such as phosphorylation, glycosylation or acylation, at specific sites on individual proteins may be detected and quantified through mass spectroscopy in accordance with the present invention.
The first sample need not contain a natural abundance of isotopes, as long as at least one isotope in the second sample of biological matter has a different abundance than the abundance of the same isotope in the first sample. Preferably, the isotope which is enriched or depleted is a non-radioactive isotope of nitrogen, oxygen, carbon and/or sulfur. Hydrogen may be used, as well. Radioactive isotopes may also be used.
The effects of two or more modulations can be simultaneously analyzed by preparing additional samples with media containing an isotope with a different abundance than the abundance of the isotope in the other samples, and modulating the additional samples.
Up to the point of the mass spectroscopy, none of the steps of the process discriminates between a protein that contains the natural abundance of isotopes from the same protein from the enriched sample. Thus, the ratios of the original amounts of proteins from the two samples are maintained, normalizing for differences between extraction and separation of the proteins in the samples.
The method is applicable to the components of any type of biological matter which can be ionized and therefore may be analyzed by mass spectroscopy. For example, the component may be a protein, a peptide, a carbohydrate, a lipid, a cofactor and post-synthetic derivatives thereof. The biological matter may be a culture of biological cells, a microbiological culture, biological tissue, an organ, an organism, a collection of organisms, a part of an organism, and a cell-free biological mimetic system, for example.